FR2649122A1 - IMPROVEMENTS IN CHAIN POLYMERIZATION REACTION AMPLIFICATION (PCR) TECHNIQUES - Google Patents

Abstract

The method of detecting and / or identifying a nucleic acid sequence or a mixture of nucleic acid sequences by enzymatic amplification and hybridization comprises the following steps: (1) a step of destroying the 5 'ends double-stranded nucleic acid sequences to prevent exponential amplification of contaminating double-stranded sequences (previously amplified sequences), by contacting an appropriate 5 'exonuclease active at a temperature between 37 and 42degree (s) (thermosensitive) ; (2) an amplification step proper by bringing the sample obtained in 1 into contact with the appropriate PCR reagents, in particular primers suitable for detection, in the presence of a heat-resistant DNA polymerase; (3) a step of detecting the specific amplified sequences.

Description

The present invention relates to a person

  improvement of nucleic acid amplification techniques by polymerization reaction in

chain (PCR).

  Polymer-ion chain reaction (PCR) was developed by SAIKI R. et al. (Science, 1985, 230, 1350). This technique makes it possible in particular to amplify single-stranded or double-stranded DNA sequences and is based on

  on the cyclic functioning of a DNA polymerase,

  which enzyme is capable of copying a strand of DNA, used

  as a matrix, into a complementary strand by elongation

  from the free 3 'OH end of an oligonucleotide primer. The PCR technique consists in carrying out n successive amplification cycles, during which two primers direct the amplification of the double-stranded DNA sequence that they frame. An amplification cycle

  consists of three steps to achieve suc-

  the denaturation of the DNA (at a temperature between 90 and 95 ° C.), the hybridization of the primers (for example at a temperature between 37 and 75 ° C.)

  and elongation of the DNA strands by DNA polymerase.

  PCR makes it possible to amplify sequences

  of nucleic acids thousands of times.

  This amplification technique is more

  specifically described in the European Patent Application CETUS 201 184, which specifies in particular that the two oligonucleotide primers used in the PCR

  are preferably single-stranded and must be sufficiently

  long time to begin the synthesis of the product

  extension in the presence of DNA polymerase.

  To avoid the addition of enzymes at each cycle, a DNA polymerase capable of withstanding 100 ° C, the

  Taq DNA polymerase, described in the Patent Application

  CETUS 258 017, is added from the beginning of the process and allows up to 40 consecutive amplification cycles. Taq polymerase allowed on the one hand to automate the amplification process and on the other hand

  to increase the specificity of the amplification.

  The amplified sequences are then detected

  FOA; several detection techniques have been proposed. Hybridization can be mentioned in particular

  between a detection probe and the nucleic acid sequence

  amplified target key, on a solid support (dot blot, Southern blot), as described in the European Patent Application CETUS 200 362 which describes the amplification technique, associated with a hybridization method,

  for the detection of a nucleic acid sequence. -

  European Patent Application CETUS 229,701 describes a method of detecting viruses by amplification

  and hybridization either on solid support or in solution.

  In the latter case, for example, the nucleic acid sequence

  key hybridized and hybridized in solution to a labeled oligonucleotide probe, specifically hybridizes to the

  target sequence, said probe being detected by the

  oligomeric restriction. The probe fragment

  labeled cleavage of the restriction enzyme

  propriety is separated on electrophoresis gel and gel

is autoradiographed.

  These two European applications do not prevent false positives due to target fragments

  previously amplified so-called contagious fragments

  nants. In fact, while the contaminations by

  homologous events, cellular debris or plasmids may

  should be avoided in laboratories dedicated to

  diagnosis, contamination due to the presence of

  specific quenches to be detected in the majority of the cases, of previous amplifications, which generate a large number of copies and cause an accumulation of these amplified target fragments, is particularly difficult to avoid and results in particular in the presence

  ubiquitous sequences & detect in the lab.

  As stated by Y.A.D. LO et al., In

  Lancet, 1988, ii, 679, this contamination is a

  reveal the high sensitivity of PCR. LO et al. describe more particularly an example of contamination in a diagnosis of HBV sequences and show a

  positive result obtained from negative samples

  HBV sequences were amplified from negative samples, a verification of the absence of

  sequences to be detected in the sample that has been

by a Southern Blot.

  LO et al. analyzed this result as a contamination of the primers used by plasmids containing the HBV insert (plasmid pHBV130), because when we get rid of the contaminated primers, there is more

  false positive amplifications in the samples

negative ions.

  LO et al. suggest that, since PCR is increasingly used in diagnosis, the

  problem of false positives, by contamination with

  identical to those to be detected, can not be

  neglects. LO et al. also suggest systematic realization

  only a so-called negative control consisting solely of reagents for PCR to assess the contamination

  possible plasmid of these reagents.

  LO et al. also propose to check

  the existence of plasmid contamination by amplification

  cation in the presence of primers placed on both sides

  of the vector-insert junction of said plasmid. This pro-

  cedure is especially necessary if results

  conflicts are obtained by conventional methods

such as Southern Blot.

  The precautions suggested above by LO et al. avoid possible contamination

  plasmid PCR reagents. It should be noted, however,

  as the problem of contamination by the presence of a plasmid vector of the target fragment arises only

  in the research laboratories, while the laboratories

  diagnostic tests (medical analysis laboratories for example), performing routine identifications, have ready-to-use reagents and in this case it is important to avoid contamination of the matrix by products other than plasmids (PCR products, free viruses, for example) for reliable diagnosis, as this contamination can not be

  avoided. in the opinion of LO et al, at present.

  That is why, the Claimant has given

  purpose of providing a polymer reaction process

  modified chain reaction (PCR), which better meets the needs of the practice than the previous methods, in particular in that it makes it possible to prevent amplification

  exponential, double-stranded nucleic acid

  which are sequences identical to those

  amplified detections during previous diagnosis

  in the laboratory, which could lead to

false positives.

  The present invention relates to a method for detecting and / or identifying a nucleic acid sequence or a mixture of nucleic acid sequences by enzymatic amplification and hybridization, characterized in that said detection comprises the steps following: (1) a step of destroying the ends of the double-stranded nucleic acid sequences to prevent the exponential amplification of contaminating double-stranded sequences (previously amplified sequences) by contact with an appropriate exonuclease

  active at a temperature between 37 and 42

  mosensible); (2) an actual amplification step by contacting the sample obtained in (1) with the appropriate reagents of the PCR, in particular primers

  appropriate for detection, in the presence of a polymeric DNA

heat-resistant rase;

  (3) a step of detecting the particular sequences

amplified.

  According to an advantageous embodiment of the invention, step (2) of said process is carried out, in

  in addition, in the presence of a thermosensitive DNA polymerase.

  According to another embodiment of implementation

  of the invention, the 5 'exonuclease is the 5' lambda

exonuclease.

  According to an advantageous arrangement of this embodiment, the primers used for

  amplification are phosphorylated at their 5 'ends.

  According to another embodiment of implementation

  said process, step (1), namely the step of

  struction of the 5 'ends of the nucleic acid sequences

  double-stranded key, is only performed once while

  step (2) is repeated at least once.

  Such a method has the advantage of modifying, at the beginning of the reaction, the amplified target fragments during

  previous manipulations (contaminants), without modification

  important features of plasmid or genomic DNA.

  After this treatment, the pre-amplified amplified target fragment

  can no longer serve as a matrix for an amplification

  exponentially, while plasmid or genomic DNA

  still remains the starting matrix.

  The 5 'exonuclease destroys the 5' ends of double-stranded DNAs as primer extension products, whereas single-stranded DNA, primers and

  denatured DNA are not destroyed by the 5 'exonuclear DNA.

  Clease. The present invention also relates to an enzymatic composition for the implementation of a detection method according to the invention, characterized in that it comprises a 5 'thermosensitive exonuclease

  and a heat-resistant DNA polymerase.

  According to an advantageous embodiment of said composition, it further comprises a DNA

  active polymerase at 37-42 ° C (thermally sensitive).

  The present invention further relates to a box, kit or set coordinated, ready for use, for carrying out the method according to the invention, characterized in that it comprises in addition to the useful amounts of buffers and suitable reagents, at least one suitable primer pair and optionally at least one

  appropriate probe, appropriate doses of a

  Enzymatic composition comprising a 5 'exonuclease, a heat-resistant DNA polymerase and optionally a DNA

active polymerase at 37-42 ° C.

  In addition to the foregoing, the invention also includes other provisions, which

  will emerge from the description which will follow, which

  give examples of the implementation of the process which is

the present invention.

  It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not constitute any

way a limitation.

  In general, the process according to the invention makes it possible to eliminate false positives due to contamination by sequences identical to the sequences

  to detect, previously amplified in the laboratory.

  In conventional amplification, as described in the CETUS patent applications for example, the contaminating sequences most of the time present in the laboratory, are in fact amplified exponentially.

  and lead to the obtaining of false positives, as

sible in Figure 1.

  On the other hand, when implementing the pro-

  According to the invention, the contaminating sequences in the presence of 5 'exonuclease do not lead to

  false positives, because they are only

  only linearly as shown in FIG. 2; in fact, the ends of the amplified fragments are very important for the hybridizations with the primers and the 5 'ends can not be repaired by the 5'-3' polymerase: the specific destruction of the extremities

  5 'previously amplified fragments (contami-

  nants), no longer allows them to be at the origin of a

exponential amplification.

  ExmpII: 1 Detection of Chlamydia trachoma

  by the process according to the invention, in the presence

  of heat-resistant DNA polymerase.

  Chlamydia DNA samples are often

  amplification in a reaction medium containing

  in a total volume of 50 μl: 1 MM of each of the appropriate 5'-phosphorylated primers, 200 μM of each of the 4 deoxyribonucleotide triphosphates (dATP, dCTP,

  dGTP, dTTP), 10 mM Tris, pH 8, 50 mM KCl, 5 mM

  MgCl2, 1 unit of 5'3 'heat-resistant polymerase and a

  unit of 5 'lambda exonuclease thermosensitive.

  20.Le. protocol is as follows: the samples are heated to 37 ° C

  minutes (exonuclease action) and are then

  increased to 25 cycles as follows:

  firing at 92 ° C for 15 seconds, cooling to 55 ° C

  for one minute and then heated to 72 C for half a minute

  nute. Samples are then stored at 72 ° C

10 minutes.

  The products of the PCR are separated by

  trophoresis on agarose gel (1.4%) bromide stained

ethidium.

  The DNA is then detected by exposure to

UV rays..

  Figure 3 shows the result obtained in

  2. A task that corresponds to the pre-

of Chlamydia DNA.

  ! x * mIe 2: Detection of Chlaaydia by ampli

classic fication.

  The procedure is as in Example 1. However, no lambda exonuclease is added. Figure 3 shows the result obtained in column 1. It is therefore seen that the presence of 5 'lambda

  exonuclease does not affect the amplification itself.

  xezffln 3: Highlighting the role of the

  contamination by sequences identical to those

  in the diagnosis of false positives: role of the

process according to the invention.

  1. Amplification of possible contaminants to be able to visualize them in agarose gel, in the presence

phosphorylated primers.

  A reaction medium containing a total volume of 50 41 μM of each of the appropriate primers for Chlamydia, phosphorylated 5 ', 200 μM each of the four deoxyribonucleotide triphosphates, mM Tris, pH 8, 50 mM was prepared. KCl, 5 mM of MgCl 2 and one

  unit of 5'3 'heat-shrink polymerase.

  Samples are subject to amplification

  25 cycles as follows: heating at 92 ° C for 1 second, cooling to 55 ° C for 1 minute,

  then heating at 72 C for one minute. Samples

  lons are then stored at 72 ° C. for 10 minutes.

  - Take 10 μl of the amplified sample

  obtained and the procedure is as in example i (see

Figure 3, column 4).

  - At the same time, a control without

  addition of exonuclease (Figure 3, column 3).

  Figure 3 shows the results obtained,

  lanes 3 and 4: column 3 corresponds to conventional amplification (without 5 'exonuclease): a slight band is observed corresponding to the specific amplified fragments of Chlamydia ,. while the starting sample does not contain Chlamydia. There is another band below this band corresponding to non-fragment

  specificities, usually found during amplification

  conducted in the absence of any DNA template; column 4 corresponds to an amplification according to the present invention (in the presence of exonuclease): no specific band of Chlamydia is observed; only the band corresponding to non-specific fragments, usually found during amplifications conducted in the absence of any

DNA template is observed.

  2. Amplification of possible contaminants in

  primer presences suitable for Chlamydia, non-phosphorylated

Rylees.

We proceed as in 1 above.

  Figure 3 shows the results obtained in columns 5 and 6:

  column 5, qi corresponds to an amplification

  conventional cation (without 5 'exonuclease) contains two bands as in column 3; column 6 is identical to column 5:

  we see that when the primers are not phosphorylated

  exonuclease is significantly less sensitive and does not

  does not systematically eliminate contaminants.

  Example 4: Detection of Chlamydia trachoma

  by the process according to the invention, in the presence

  of a heat-resistant DNA polymerase and a poly-

thermosensitive merase.

  The reaction is carried out as follows:

  DNA samples denatured by the chain

  are added to a buffer containing 10 mM Tris, pH 8, mM KCl, 5 mM MgCl 2, dXTP: 200 M each, primer: 1 MM each, 0.5 unit lambda DNA exonuclease, 0.25 unit of active DNA polymerase at 37-42 ° C (transcriptase

  reverse) and 0.5 units of heat-resistant DNA polymerase.

  The samples are heated at 37 ° C for minutes and undergo a 25-cycle amplification

  as follows: the samples are heated to 90 ° C

  15 seconds, cooled to 55 ° C for one minute and heated to 72 ° C for one minute. The samples are

  then stored at 72 ° C for 10 minutes.

  The PCR products are separated on agarose gel stained with ethidium bromide. The DNA is then detected by exposure to U.V. rays. The Southern tranferts are carried out on Hybond N (Amersham). 32p-labeled, single-stranded RNA specific for sequences

  Amplified targets is hybridized to the filter.

  As is apparent from the foregoing, the invention is in no way limited to those of its modes of

  Implementation, implementation and application which

  to be described more explicitly; she

  on the contrary, all variants that can

  to the technician's mind in this respect, without

  the scope and scope of the present invention.

11.

NEEDICMTIOHS

  1.) A method for detecting and / or identifying a nucleic acid sequence or a

  mixture of nucleic acid sequences by amplification

  enzyme detection and hybridization, characterized in that said detection comprises the following steps: (1) a step of destroying the ends of the double-stranded nucleic acid sequences to prevent the exponential amplification of contaminating double-stranded sequences (previously amplified) by contact with an appropriate exonuclease

  active at a temperature between 37 and 42 '(thermal

  mosensible); (2) an actual amplification step by contacting the sample obtained in (1) with the appropriate reagents of the PCR, in particular primers

  appropriate for detection, in the presence of a polymeric DNA

heat-resistant rase;

  (3) a step of detecting the particular sequences

amplified.

  2 ') Process according to claim 1, characterized

  in that step (2) of said method is carried out,

  in addition, in the presence of a thermosen DNA polymerase

  sible. 3 ') Method according to claim 1 or claim 2, characterized in that the 5' exonuclease

is the 5 'lambda exonuclease.

  4 ') Process according to any one of the

  1 to 3, characterized in that the primers used for the amplification are advantageously

  phosphorylated at their 5 'ends.

  Process according to any one of the

  1 to 4, characterized in that step (1), at

  see the step of destruction of the 5 'ends of the

  double-stranded nucleic acid sequences, is performed only once while step (2) is repeated at least

Once.

  6 ') Enzyme composition for carrying out a detection method according to any one of

  Claims 1 to 5, characterized in that

  comprises a thermosensitive exonuclease and a po-

thermoresistant lymerase.

  7) Composition according to claim 7,

  characterized in that it further comprises a DNA

  active polymerase at 37-42 ° C (thermally sensitive).

  8 ') set, kit or set coordinated, ready to use, for the implementation of the method according to one

  any of claims i to 5, characterized in that

  that it understands off the useful amounts of buffers and

  suitable reagents, at least one pair of primers

* propriety and possibly of at least one probe appro-

  appropriate doses of an enzymatic composition.

  comprising a 5 'exonuclease, a DNA polymerase

  heat-resistant and possibly an active DNA polymerase

at 37-42C.